Despite the tremendous success of vaccines in limiting the spread of infectious diseases, there remains a large unmet need for development of vaccines against major human pathogens such as HIV. Within this context, replication-deficient type 5 adenovirus vector (AdV) vaccines expressing target antigens have received considerable attention because of their relative safety and ability to induce humoral and cell-mediated responses. Host responses against infectious agents or vaccines require highly conserved innate immunity-inducing pattern recognition receptors (PRR), including Toll-like Receptors (TLRs) and RIG-I-like receptors. Despite the presence of multiple PRR, only a few intracellular pathways are thought to be critically important for induction of host responses by PRR. Key amongst them are transcription factors belonging to the NF-kB and IRF families. These transcription factors therefore serve as intracellular adjuvants in promoting immune responses. The main goal of studies proposed here is to determine whether the vaccine potential of AdV can be enhanced through expression of key intracellular adjuvants. To this end, the studies proposed here will systematically evaluate the adjuvant potential and mechanism of action of different key molecules that activate NF-kB and IRF pathways. If successful, our studies can have a major impact on the future design of AdV and potentially non-AdV vaccines for both infectious diseases and cancer. Two specific aims are proposed. Aim 1: Generation, in vitro and in vivo characterization of AdV expressing distinct intracellular adjuvants. Aim 2: Defining the role of type 1 IFN in intracellular adjuvant-induced enhancement of AdV immune responses.